Interpretive Summary: We previously demonstrated transient expression of exogenous genes in Babesia bovis after electroporation of intraerythrocytic B. bovis stages with plasmids containing the luciferase gene under the control of Babesia bovis promoters. However, attempts to establish stable transfection of B. bovis using the same electroporation protocol have so far been unsuccessful. Since Babesia merozoites are obligatory intracellular organisms that reside inside erythrocytes, a successful transfection system must be capable of transferring exogenous DNA through the erythrocyte, merozoite and nuclear membrane layers. As a result, stable transfection can be severely limited by low transfection efficiency. In this study, we describe the development and optimization of methods for transfection of purified B. bovis merozoites using either nucleofection (Amaxa) or conventional electroporation (Gene Pulser II, BioRad). Overall, the results provide a basis for optimal transfection of purified B. bovis merozoites using either nucleofection or conventional electroporation. However, nucleofection is significantly more efficient when transfecting either circular or restriction digested DNA in the 2-10 micrograms range.

Technical Abstract:
Transient transfection of intraerythrocytic Babesia bovis parasites has been previously reported. In this study, we describe the development and optimization of methods for transfection of purified B. bovis merozoites using either nucleofection (Amaxa) or conventional electroporation (Gene Pulser II, BioRad). Initially, the optimal buffer (“Plasmodium 88A6”) and program (v-24) for nucleofection of free merozoites with a plasmid containing the luciferase gene as a reporter were determined. Using the same reporter plasmid, optimal voltage, capacitance and resistance for transfecting free merozoites by electroporation were defined to be 1.2 kV/25 microF/200 ohms. Using these optimal parameters, analysis of the time course of luciferase expression using either system to transfect free B. bovis merozoites showed a peak at 24 hours, with a rapid decline thereafter. Nucleofection was approximately five times more effective than electroporation when using a small quantity (2 micrograms) of DNA, while electroporation was twice as effective as nucleofection when a larger quantity of plasmid DNA (100 micrograms) was used. Comparison of luciferase expression after transfection of merozoites with circular, linearized, or double digested plasmid indicated that intact, circular plasmid was necessary for optimal luciferase expression. Overall, the results provide a basis for optimal transfection of purified B. bovis merozoites using either nucleofection or conventional electroporation. However, nucleofection is significantly more efficient when transfecting either circular or restriction digested DNA in the 2-10 micrograms range.